Project Summary/ Abstract Driver mutations enhance the growth, invasion and metastasis of melanoma. In addition to oncogenic drivers, melanoma escape immune recognition by activation of inhibitory immune checkpoint pathways. Based on this knowledge, treatment of melanoma with inhibitors of activated BRAF plus downstream MEK have shown efficacy and survival benefits in metastatic disease. Immune checkpoint inhibitors have also demonstrated striking efficacy in melanoma and the combination of anti-PD-1 with anti-CTLA-4 (nivolumab, nivo; ipilimimab, ipi) produces tumor responses in more than half of melanoma patients. However, drug resistant subpopulations emerge in a relatively short time after BRAF/MEK inhibitor treatment and result in disease relapse. Immune checkpoint blockade frequently causes immune-related Adverse Effects (irAEs) due to the disruption of the self-tolerance of normal tissues requiring immunosuppressive treatment. It is unclear to what extent immunosuppressive treatment to control irAEs, may compromise the anti-tumor activity of the immune checkpoint blockade. Thus, there is an unmet need for real-time detection of tumor response and resistance as well as the onset of treatment-related irAEs. Here I propose to monitor anti-tumor efficacy as well as organ- specific irAEs using cell-free DNA analysis from serial blood samples obtained before and at regular intervals during and after treatment of patients with BRAF mutant metastatic melanoma. In a randomized trial patients first receive either a BRAF/MEK inhibitor combination or nivo/ipi and are switched to the other arm upon disease recurrence or toxicity. Serial blood samples are collected at defined intervals. Under Aim 1, I propose to monitor changes in circulating cell-free mutant DNA patterns as a readout of anti-tumor treatment efficacy and resistance. Under Aim 2, I propose to assess autoimmune organ damage by monitoring changes in the abundance of circulating cell-free tissue-specific methylated DNA. The readouts from the changes in these molecular markers will be compared with clinical observations in the trial. The studies will establish non- invasive detection of molecular changes during treatment that indicate incipient drug resistance and irAEs.